Following obtaining support from the Director, NCI, Dr. Waldmann directed an inter-institute collaboration (NCI-NIAID) directed toward the production under current good manufacturing practices (cGMP) of IL-15 for clinical trials in patients with metastatic malignant melanoma and renal cell cancer as well as in patients with HIV/AIDS receiving HAART therapy. In a modification of an expression system and purification method developed in the Waldmann Laboratory, the Biopharmaceutical Development Program (BDP) of the National Cancer Institute under the direction of Steven Creekmore has produced recombinant human IL-15 (rhIL-15) under current good manufacturing practices (cGMP). In this work, an E. coli based fermentation and purification process was developed for the production of clinical grade recombinant human IL-15. DNA sequences from human IL-15 were inserted into pET-28b plasmid, were expressed in the E. coli host BL21-AL. The inclusion bodies of IL-15 produced in E. coli were solubilized, refolded and orthogonally purified to yield active IL-15. RhIL-15 was produced as a non-glyclosylated, single-chain polypeptide of 115 amino acids, having a calculated molecular weight of 12,901 Da. Purified rhIL-15 showed in vitro activity in a CTLL-2 cell proliferation assay in the range of 15-25 x 106 IU/mg protein standardized against the international standard obtained from the National Institute of Biological Standard and Control (NIBSC). In a toxicology study directed by the Waldmann Laboratory, the rhIL-15 was evaluated for safety (toxicity), pharmacokinetics, immunogenicity, autoimmunity and impact on elements of the normal immune system of rhIL-15 in rhesus macaques. The only biologically meaninful laboratory abnormally was a grade 3/4 neutropenia that was secondary to a redistribution of neutrophils from the circulation to the tissues. No abnormalities were observed following IL-15 administration in gross or microscopic analysis of tissues at autopsy. The animals did not produce antibodies to IL-15 as assessed using a sensitive 2-armed capture technique developed in the Waldmann Laboratory. IL-15 administration was associated with four to eight fold increases in the number of circulating NK and CD3 CD8 especially central and effector memory T-lymphocytes. The administration was also associated with a tenfold increase in the circulating IL-2R alpha levels. CDER FDA and IRB NCI approval has been granted to initiate a clinical trial Phase I Study of Intravenous Recombinant Human IL-15 (rhIL-15) in Adults with Refractory Metastatic Malignant Melanoma and Metastatic Renal Cell Cancer. The sponsor is the Metabolism Branch, CCR, NCI with Dr. Thomas A. Waldmann as Study Chair and IND holder. The study will be performed on the Metabolism Branch, CCR, NCI in the Clinical Center with Dr. John C. Morris as the principal investigator (PI). The primary objective of this trial is to determine the safety, toxicity profile, dose-limiting toxicity and maximum tolerated dose of intravenous recombinant human IL-15 produced by the BDP and administered as a daily intravenous infusion for 12 days in individuals with incurable metastatic malignant melanoma and renal cell cancer. The secondary objectives include determination of rhIL-15 pharmacokinetics and to determine the immunogenicity of the IL-15 in individuals receiving this drug. Phenotypic analysis of circulating lymphocytes will be used to define the biological effects of rhIL-15. A final objective is to obtain preliminary information on the efficacy of IL-15 in the treatment of patients with metastatic malignancy. Following the completion of the phase I trial in patients with metastatic malignancy, phase I/II trials will be initiated in collaboration with Dr. Clifford Lane of NIAID for the evaluation of rhIL-15 in the treatment of patients with HIV/AIDS receiving HAART therapy. It is hoped that IL-15 administration will be safe and show efficacy in the treatment of metastatic malignancy in human clinical trials. However, it probably will not be optimal when used as monotherapy. For its long-term persistence and its optimal activation of NK and CD8+ T-cells, IL-15 must be bound to IL-15R alpha on the surface of DCs where it is transpresented to effector NK and CD8 T-cells. However, there is only low level expression of IL-15R alpha on resting DCs. We are examining the use of agents that induce cell surface expression of IL-15R alpha. The stimulation of monocytes or DCs with ligation of CD40 with an agonistic agent (e.g., CD40 ligand or agonistic anti-CD40 antibodies) induced the expression of IL-15R alpha. We evaluated the combination of IL-15 with an agonistic anti-CD40 monoclonal antibody in murine models of colon and prostate cancer. We demonstrated that administration of the anti-CD40 monoclonal antibody FGK4.5 was associated with an increased expression of IL-15R alpha on CD11 positive DCs present in the spleens of treated mice. Furthermore, the serum concentrations of mIL-15R alpha as assessed by ELISA remarkably increased in anti-CD40 monoclonal antibody treated mice when compared to control mice. The therapeutic efficacy of the combination regimen of murine IL-15 with an anti-mouse CD40 monoclonal antibody when evaluated in models involving CT26 and MC38 colon carcinoma administration led to a prolonged (p less than 0.01) survival of both CT26 and MC38 tumor-bearing mice when compared to mice in the PBS control group. In the TRAMP C2 model the combination therapy with both mIL-15 and anti-CD40 antibody also provided great augmentation of therapeutic efficacies as demonstrated by the fact that 80% of the TRAMP C2 tumor-bearing mice in the combination group were cured whereas only 10% of the anti-CD40 group alone and none in the mIL-15 group became tumor free. Furthermore, we demonstrated that the combination regimen significantly increased the number of tetramer positive CD8 tumor antigen specific T-cells in spleens of the TRAMP C2-bearing mice and that these mice with tetramer positive CD8 T-cells were resistant to tumor rechallenge. Cytotoxic assay of NK cells primed ex vivo by wild-type or IL-15R alpha-/- BMDCs demonstrated the expression of IL-15R alpha by DCs was critical for the synergistic killing activity of NK cells mediated by the combination regimen. These findings provide the scientific basis for clinical trials using a combination regimen involving IL-15 given in association with an agonistic anti-CD40 antibody for the treatment of patients with metastatic malignancy. Another impediment to IL-15 monotherapy is that IL-15 administration leads to the expression of certain checkpoints on the immune system. In particular, IL-15 administration led to the up-regulation of PD-1 on the tumor cells and to the release of the immunosuppressive cytokine IL-10. In addressing this issue we demonstrated that in the MC38 and TRAMP C2 murine model systems that the co-administration of antibodies to cytotoxic T-lymphocyte antigen 4 (CTLA-4) and to program cell death 1 (PD-1/PD-L1) proteins that mediate negative T-cell signaling pathways markedly augmented the efficacy of murine mIL-15 in tumor treatment. In each model the simultaneous administration of antibodies to both checkpoints was required to augment IL-15 action. These studies support a clinical trial involving rhIL-15 given in combination with antibodies to CTLA-4 and to PD-L1 in patients with metastatic malignant melanoma and metastatic renal cell cancer. In summary the studies in the Waldmann Laboratory support clinical trials that involve rhIL-15 alone as well as rhIL-15 given in conjunction with agonistic antibodies to CD40 to augment the expression of IL-15R alpha and antibodies to CTLA-4 and to PD-L1 to remove immunological checkpoints on the immune system.